Diol dehydratase-reactivating factor is a reactivase – evidence for multiple turnovers and subunit swapping with diol dehydratase Koichi Mori, Yasuhiro Hosokawa, Toshiyuki Yoshinaga and Tetsuo Toraya Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Japan Keywords adenosylcobalamin; coenzyme B12; diol dehydratase; diol dehydratase-reactivating factor; reactivase Correspondence T Toraya, Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan Fax: +81 86 251 8264 Tel: +81 86 251 8194 E-mail: toraya@cc.okayama-u.ac.jp (Received 26 July 2010, revised 17 September 2010, accepted October 2010) doi:10.1111/j.1742-4658.2010.07898.x Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co–C bond The damaged cofactor remains tightly bound to the active site The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg2+ Therefore, we concluded that the reactivating factor is a sort of enzyme It can be designated DD reactivase The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzymcyanocobalamin complex This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase The specificities of the reactivase for divalent metal ions were also not strict DD formed : and : complexes with the reactivase in the presence of ADP and Mg2+ Upon complex formation, one b subunit was released from the (ab)2 tetramer of the reactivase This result, together with the similarity in amino acid sequences and folds between the DD b subunit and the reactivase b subunit, suggests that subunit displacement or swapping takes place upon formation of the enzymreactivase complex This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD Structured digital abstract l MINT-7997177: Reactivase alpha (uniprotkb:O68195), Reactivase beta (uniprotkb:O68196), Diol Dehydratase gamma (uniprotkb:Q59472), Diol Dehydratase beta (uniprotkb:Q59471) and Diol Dehydratase alpha (uniprotkb:Q59470) physically interact (MI:0915) by comigration in non denaturing gel electrophoresis (MI:0404) l MINT-7997157: Diol Dehydratase alpha (uniprotkb:Q59470), Diol Dehydratase beta (uniprotkb:Q59471), Diol Dehydratase gamma (uniprotkb:Q59472), Reactivase beta (uniprotkb: O68196) and Reactivase alpha (uniprotkb:O68195) physically interact (MI:0915) by molecular sieving (MI:0071) Abbreviations AdePeCbl, adeninylpentylcobalamin; AdoCbl, adenosylcobalamin or coenzyme B12; CN-Cbl, cyanocobalamin; DD, diol dehydratase FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS 4931 Reactivase for coenzyme B12-dependent enzyme K Mori et al Introduction Adenosylcobalamin (AdoCbl)-dependent enzymes catalyze chemically difficult reactions by the use of highly reactive radicals The homolytic cleavage of the Co–C bond of the coenzyme forms a Co(II) species and an adenosyl radical, which triggers the reactions [1] Although enzymes generally deal with highly reactive intermediates by ‘negative catalysis’ [2], cobalamin enzymes tend to undergo mechanism-based inactivation because of the involvement of highly reactive radical intermediates during catalysis [3] Diol dehydratase (DD) (EC 4.2.1.28) catalyzes the AdoCbl-dependent conversion of 1,2-propanediol, glycerol and 1,2-ethanediol to the corresponding aldehydes [4,5] Its physiological substrates are 1,2-diols, such as 1,2-propanediol [6,7], but it functionally substitutes for glycerol dehydratase (EC 4.2.1.30), an isofunctional enzyme of DD, in the anaerobic dissimilation of glycerol by Klebsiella oxytoca and some other bacteria that lack glycerol dehydratase [8,9] Despite their roles, both enzymes undergo mechanism-based inactivation by glycerol [5,10–12], accompanying the irreversible cleavage of the Co–C bond of the enzyme-bound coenzyme The damaged cofactor thus formed remains tightly bound to the apoenzyme and is not displaced by intact AdoCbl, resulting in the irreversible inactivation of the enzyme This apparent inconsistency was resolved by our finding of the rapid reactivation of glycerol-inactivated enzymes in permeabilized Klebsiella pneumoniae and K oxytoca cells (in situ) [13,14] Specific protein factors that are responsible for the reactivation of the inactivated holoenzymes of DD [15–17] and glycerol dehydratase [18–20] were found, and designated DD-reactivating factor and glycerol dehydratasereactivating factor, respectively They reactivated the O2-inactivated [16,19,20] and 3-butene-1,2-diol-inactivated [21,22] holoenzymes as well We demonstrated that these factors reactivate the inactivated holoenzymes by a molecular chaperone-like mechanism (Fig 1) [16,17,19,23] Salient features are as follows The reactivating factor binds ATP and hydrolyzes it to ADP by its own weak ATPase activity The resulting ADP-bound form of the reactivating factor has a high affinity for the enzyme, and interacts with the inactivated holoenzyme to form a tight apoenzymreactivating factor complex, with the concomitant release of the damaged cofactor The reactivating factor reverts to a low-affinity form through the replacement of bound ADP by free ATP, resulting in the dissociation of the apoenzymreactivating factor complex into apoenzyme and the reactivating factor Active holoenzyme is then 4932 Fig Mechanism of the reactivation of inactivated holoenzymes by reactivating factors E, DD or glycerol dehydratase; RF, DD-reactivating factor or glycerol dehydratase-reactivating factor; X-Cbl, a damaged cofactor; AdoH, 5¢-deoxyadenosine reconstituted from apoenzyme and free AdoCbl DD does not form a complex with the reactivating factor while it exists as an active holoenzyme The glycerol dehydratase-reactivating factor reactivates the inactivated hologlycerol dehydratase in a similar manner Both dehydratase-reactivating factors exist as a2b2 heterotetramers [a, DdrA or GdrA (DhaF); b, DdrB or GdrB (DhaG)] [16,19,20] Liao et al reported the crystal structure of the nucleotide-free form of glycerol dehydratase-reactivating factor [24] Independently, we solved the crystal structures of the DD-reactivating factor in both the ADP-bound and nucleotide-free forms [25] The structures of both reactivating factors are similar Their a subunits have a structural feature common to the ATPase domains of actin superfamily proteins, including Hsp70 molecular chaperones Interestingly, their b subunits have similar folds to the b subunits of diol and glycerol dehydratases Such structural characteristics provide important clues to help solve the mechanisms of action of these reactivating factors – that is, subunit swapping might occur However, no biochemical evidence for this has been obtained so far A similar reactivating factor for ethanolamine ammonia lyase has been reported [26] It has also been reported that a protein named E2 activates lysine-5,6-aminomutase in an ATP-dependent manner, although its exact function is not yet known [27] In this study, we examined whether and how the complexes between DD and its reactivating factor are formed Specificities of the reactivating factor for nucleotides and divalent cations were also investigated In addition, it was determined whether the reactivating factor-mediated cobalamin release is catalytic FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS Reactivase for coenzyme B12-dependent enzyme K Mori et al Results A Evidence for multiple turnovers of DD-reactivating factor in cobalamin exchange In a previous article, we reported the number of the reactivating factor-mediated reactivations of DD during the dehydration of glycerol [16] The number of reactivations per molecule of DD was calculated to be approximately six under conditions where the reactivating factor was added to a 10-fold molar excess relative to the enzyme This indicates that the enzyme undergoes multiple inactivation–reactivation cycles On the other hand, the maximum number of reactivations per molecule of the reactivating factor was observed to be approximately two, at a molar ratio of the reactivating factor to the enzyme of 0.5 As the reactivating factor exists as a dimer of ab heterodimers, i.e (ab)2, it remained unclear whether the reactivating factormediated reactivation of inactivated holoenzymes is catalytic or stoichiometric It is experimentally not possible to demonstrate multiple turnovers for the reactivating factor in this reactivation assay, probably because of the inhibition of the holoenzyme by accumulated 3-hydroxypropionaldehyde To avoid this difficulty, we examined whether the reactivating factor can mediate multiple turnovers of the replacement of tightly bound cyanocobalamin (CN-Cbl) (an inactive coenzyme analog lacking the adenine ring in the upper axial ligand; a model of damaged cofactors) for free adeninylpentylcobalamin (AdePeCbl) (an inactive coenzyme analog containing the adenine ring in the upper axial ligand; a model of intact coenzyme, AdoCbl) in the presence of ATP and Mg2+ at a molar ratio of the reactivating factor to the enzyme of 0.1 (Fig 2) The spectrum of the enzyme obtained after the removal of unbound cobalamins indicated that the enzyme-bound CN-Cbl was replaced by AdePeCbl in a manner dependent on both the reactivating factor and ATP ⁄ Mg2+ (Fig 2A,B) Figure 2C shows the time course of the exchange of enzyme-bound CN-Cbl for AdePeCbl in the presence of the reactivating factor and ATP ⁄ Mg2+ About 70% of the enzyme-bound CN-Cbl was replaced with AdePeCbl within h, and the replacement was almost complete within h The total amount of enzyme-bound cobalamin remained almost constant (1.9–2.1 molỈmol)1) during incubation It is thus evident that the reactivating factor mediates the exchange of enzyme-bound CN-Cbl for free AdePeCbl for a 10-fold molar excess of the enzyme under the conditions employed This strongly suggests that the reactivation of the inactivated holoenzymes by the factor is not stoichiometric but catalytic Hence, the B C Fig Evidence for the catalytic turnover of the DD-reactivating factor (A) The reactivating factor-mediated replacement of enzymebound CN-Cbl with free AdePeCbl in the presence of ATP and Mg2+ was analyzed by the spectral change of enzyme-bound cobalamin A 10-fold excess of the enzymCN-Cbl complex over the reactivating factor was used Experimental details are described in the text After removal of unbound cobalamin at (thick solid line), 30 (thin solid line), 60 (thin long-dashed line), 120 (thin short-dashed line), 240 (thin dotted line) and 360 (thick dotted line) of incubation, the spectrum of enzymebound cobalamin was measured Inset: spectra of enzyme-bound CN-Cbl (solid line) and AdePeCbl (dotted line) (B) Experimental conditions were the same as in (A), except that spectra were taken after 360 of incubation in the absence of ATP and Mg2+ (thick solid line) or without the reactivating factor in the presence of ATP and Mg2+ (thick dotted line) (C) Time course of the reactivating factor-mediated exchange of enzyme-bound CN-Cbl for AdePeCbl The extent of exchange was determined from the change in absorbance at 364 nm The total amount of enzyme-bound cobalamin was determined spectrophotometrically after conversion to the dicyano form Inset: a semilogarithmic plot FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS 4933 Reactivase for coenzyme B12-dependent enzyme K Mori et al DD-reactivating factor can be designated DD reactivase as well The cobalamin exchange occurs through the intermediary formation of apoenzyme [17,19] As AdePeCbl binding to apoenzyme takes place much faster than CN-Cbl release, the rate of CN-Cbl replacement can be considered to be the rate of CN-Cbl release The inset of Fig 2C indicates that the rate of CN-Cbl replacement (release) follows pseudo-first-order kinetics, and the rate constant of the reactivase in cobalamin release (kcat,cbl-release) for CN-Cbl was calculated to be 0.27 min)1 at 37 °C from the initial rate The time course of the reactivation of glycerol-inactivated holoenzyme by the reactivase in the presence of ATP and Mg2+ at a molar ratio of the reactivase to the enzyme of 0.1 is shown in Fig From the initial rate, the rate constant of the reactivase in the reactivation (kcat,react) was calculated to be 0.071 ± 0.008 min)1 at 37 °C Considering that the enzyme contains two cobalamin-binding sites in the (abc)2 dimer, it can be assumed that the reactivase mediates the exchange of enzyme-bound damaged cofactor for intact AdoCbl with a rate constant (kcat,cbl-release) of 0.14 min)1 Kinetic parameters of the reactivase for ATP in DD (re)activation and ATP hydrolysis Kinetic constants for ATP in the reactivation of glycerol-inactivated holoenzyme and the activation of the enzymCN-Cbl complex by the reactivase were measured (Table 1) Km values for ATP in the reactivation and the activation were essentially the same: 6.9 ± 0.4 mm and 6.8 ± 1.6 mm, respectively This is reasonable, because these two events are different aspects of the same phenomenon [14,16] Km values for the ATPase activity were also measured in the presence and absence of equimolar apoenzyme (Table 1) The Km for ATP in the ATPase activity in the absence of enzyme was 61 ± 14 lm, i.e two orders of magnitude smaller than that in the DD (re)activation Moreover, the Km for ATP in the ATPase activity was essentially not affected by the presence of enzyme (67 ± 17 lm) The kcat in the ATPase activity was estimated to be 1.4 ± 0.1 min)1 in the absence of apoenzyme The ATPase activity was slightly inhibited by the presence of apoenzyme (kcat = 1.2 ± 0.1 min)1) These values are in good agreement with those previously reported [17] Nucleotide and divalent cation specificities of the reactivase The specificities of the reactivase for nucleotides in the activation of the enzymCN-Cbl complex were studied in the presence of AdoCbl and Mg2+ (Table 2) Although ATP was most effective (40% as compared with the apoenzyme control), CTP, UTP and GTP showed comparable efficiencies (82%, 75% and 55% relative to ATP, respectively) Moreover, 2¢-dATP and 3¢-dATP were 55% and 63% as effective as ATP The efficiencies of various divalent metal ions for the activation of the enzymCN-Cbl complex by the reactivase were also examined at mm (Table 2) The Table Kinetic parameters of the reactivase for ATP Km for ATP (mM) Reactivationa Activationa ATPase (+DD)b ATPase ()DD)b Fig Time course of the reactivation of the glycerol-inactivated holoenzyme by DD reactivase The glycerol-inactivated holoenzyme formed as described in the text was subjected to ultrafiltration on a Microcon YM-10 microconcentrator (Millipore) To a concentrated protein fraction containing 1.2 nmol of glycerol-inactivated holoenzyme, we added 2.3 M 1,2-propanediol, 38 lM AdoCbl, 19 mM ATP and 19 mM MgCl2 in 0.02 M potassium phosphate buffer (pH 8.0) without or with 0.12 nmol of reactivase to a total volume of 160 lL After incubation at 37 °C for the indicated time periods, 20 lL aliquots were withdrawn, and the amount of DD reactivated was measured by the 3-methyl-2-benzothiazolinone hydrazone method [33] after appropriate dilution 4934 6.9 6.8 0.067 0.061 ± ± ± ± 0.4 1.6 0.017 0.014 Vmax (lmol propionaldehyde formed in 10 min) kcat (min)1) 2.2 ± 0.6 17 ± 1.2 ± 0.1 1.4 ± 0.1 a The glycerol-inactivated holoenzyme (31 pmol) or the enzymCNCbl complex (53 pmol) was incubated at 37 °C for 10 with 0.21 nmol of reactivase in 70 lL of 0.02 M potassium phosphate buffer (pH 8.0) containing 0.6 M 1,2-propanediol, 0.01 M KCl, and 21 lM AdoCbl, with 0–40 mM each of ATP and MgCl2 The reaction was terminated by addition of 70 lL of 0.1 M potassium citrate buffer (pH 3.6) The amount of propionaldehyde formed was determined as described in the text after appropriate dilution b The reactivase (0.22 nmol) was incubated at 37 °C for with or without apoenzyme (5.0 units, 0.20 nmol) in 50 lL of 0.01 M potassium phosphate buffer (pH 8.0) containing 0.3–10 mM each of [32P]ATP[cP] and MgCl2 ATPase activity was measured as described in the text FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS Reactivase for coenzyme B12-dependent enzyme K Mori et al Table Nucleotide and divalent cation specificities of the reactivase for the activation of the enzymCN-Cbl complex The enzymCN-Cbl complex (DDỈCN-Cbl) (61 pmol) was incubated at 37 °C for 10 with and without 0.30 nmol of reactivase in 50 lL of 0.02 M potassium phosphate buffer (pH 8.0) containing 21 lM AdoCbl and 1.2 M 1,2-propanediol in the presence and absence of mM ATP (or an indicated nucleotide) and mM MgCl2 (or a chloride salt of the indicated divalent metal ions) The reaction was terminated by addition of 50 lL of 0.1 M potassium citrate buffer (pH 3.6) The amount of propionaldehyde formed was determined as described in the text after approriate dilution Propionaldehyde formed Run no Enzyme Reactivase Nucleotide Metal ion apoDD DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl apoDD DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl DDỈCN-Cbl ) + + + + + + + ) + + + + + + + + ) ) ATP GTP CTP UTP 2¢-dATP 3¢-dATP ) ATP ATP ATP ATP ATP ATP ATP ATP ) ) Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ ) ) Mg2+ Ca2+ Cr2+a Mn2+ Co2+ Ni2+ Cu2+ 13.4 0.1 5.3 3.0 4.4 4.0 2.9 3.4 13.7 0.0 6.0 0.1 0.1 11.1 5.8 2.9 0.1 (%) (lmol) a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.6 0.1 1.2 0.4 1.0 0.8 0.6 0.1 1.8 0.0 0.2 0.1 0.1 0.4 0.1 0.7 0.0 Relative activity (%) 100 40 22 33 30 22 25 100 44 1 81 42 21 ) ) 100 57 83 75 55 64 ) ) 100 2 185 97 48 Cr2+ added might be oxidized to Cr3+ by air in the reaction mixture divalent cations tested did not inhibit the DD activity at mm in the standard assay conditions (data not shown) Mn2+ was most effective for the activation (81% as compared with the apoenzyme control; 185% relative to Mg2+) Co2+ and Ni2+ were also effective (97% and 48%, respectively, relative to Mg2+), whereas Ca2+, Cr2+ (Cr3+) and Cu2+ were not (< 2% relative to Mg2+) Mg2+ was used routinely in the (re)activation assay, because it is a physiological divalent metal ion The specificity of the reactivase for divalent metal ions in ATP hydrolysis was also measured (Table 3) Although the reactivase hydrolyzed ATP to some extent even in the absence of divalent metal ions, Mg2+, Mn2+, Co2+ and Ni2+ enhanced the ATPase activity by 3.7–4.5 fold at mm On the other hand, Ca2+, Cr2+ (Cr3+) and Cu2+ had little or no effect on the ATPase activity as compared with the control without divalent metal ions Irrespective of the presence of divalent cations, ATP was hydrolyzed to ADP + Pi by the reactivase (data not shown) Analysis of complex formation between DD and its reactivase by gel filtration Apoenzyme was incubated with the reactivase in the presence of ADP or ATP and Mg2+, and then subjected Table Divalent cation specificity of the reactivase for the ATPase activity The reactivase (0.22 nmol) was incubated at 37 °C for 30 with mM [32P]ATP[cP] in 50 lL of 0.01 M potassium phosphate buffer (pH 8.0) in the presence and absence of the indicated divalent metal chloride (3 mM) The ATPase activity was measured as described in the text Metal ion ATPase activity (min)1) Relative activity (%) Mg2+ Ca2+ Cr2+a Mn2+ Co2+ Ni2+ Cu2+ – 1.99 0.52 0.38 2.23 2.14 2.42 0.80 0.54 100 26 19 112 107 122 40 27 ± ± ± ± ± ± ± ± 0.04 0.01 0.01 0.01 0.04 0.09 0.05 0.02 a Cr2+ added might be oxidized to Cr3+ by air in the reaction mixture to gel filtration on a Superose column that had been preliminarily equilibrated with nucleotide ⁄ Mg2+containing buffer (Fig 4) In the presence of ATP, the enzyme and the reactivase eluted separately at their respective retention times In contrast, a peak of the free reactivase decreased and a new peak appeared in the presence of ADP The latter peak was eluted with a FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS 4935 Reactivase for coenzyme B12-dependent enzyme K Mori et al A B Fig Analysis of interaction between DD and its reactivase by gel filtration column chromatography Experimental details are described in the text 1, apoenzyme + reactivase (+ADP); 2, apoenzyme (+ADP); 3, reactivase (+ADP); 4, apoenzyme + reactivase (+ATP) Positions of the free apoenzyme (DD), reactivase (DD-R) and the enzymreactivase complex(es) (DDỈDD-R) are indicated on the tops of chromatograms 66 K– C retention time that was slightly shorter than that of the free enzyme and much shorter than that of the reactivase When this peak was subjected to SDS ⁄ PAGE, the peak comprised all of the subunits from the enzyme (a, b, and c) and the reactivase (a and b) (data not shown) It was thus evident that this peak contained the enzymreactivase complex(es) in addition to a small amount of the free enzyme In fact, when this peak was analyzed by nondenaturing PAGE, two bands of the enzymreactivase complexes described below were detected (data not shown) A similar peak containing the enzymreactivase complexes was observed in the absence of nucleotide as well, although the content of complexes was rather lower than in the presence of ADP Characterization of the enzymreactivase complexes by nondenaturing PAGE When the enzymCN-Cbl complex was incubated with the reactivase in the presence of ADP and Mg2+ and followed by nondenaturing PAGE, the bands of the enzyme and the reactivase were markedly reduced in density, and two bands of the enzymreactivase complexes (upper and lower) appeared above them (Fig 5A, lane c) When apoenzyme was used instead of the enzymCN-Cbl complex, both bands of the enzymreactivase complexes were observed in the presence of ADP (Fig 5A, lane e) The abundance of 4936 66 K– 45 K– 36 K– 29 K– 24 K– 20 K– 14 K– Fig Analysis of the enzymreactivase complex by PAGE Experimental details are described in the text (A) Nondenaturing PAGE in the presence of ADP and Mg2+ Lanes: (a) apoenzyme; (b) reactivase; (c) enzymCN-Cbl complex + reactivase (+ADP); (d) enzymCN-Cbl complex (+ADP); (e) apoenzyme + reactivase (+ADP); (f) apoenzyme (+ADP); (g) reactivase (+ADP) +ADP indicates that samples were incubated with ADP ⁄ MgCl2 (B, C) Bands i–vi in (A) were excised and subjected to SDS ⁄ PAGE on 6% (B) and 14% (C) gels Lanes i–vi correspond to bands i–vi in (A) Lane D: purified enzyme Lane R: purified reactivase Positions of DD and its subunits (aD, bD and cD) and the reactivase (DD-R) and its subunits (aR and bR) are indicated on the right of the gels In (C), to improve visibility of the bands of small subunits, especially bR, three excised pieces of the same bands from nondenaturing PAGE were subjected together to SDS ⁄ PAGE This resulted in saturation of the bands of large subunits, i.e aD and aR Densitometric analysis was carried out with other gels in which such saturation did not occur (not shown) the upper band relative to the lower band of the enzymreactivase complex formed from apoenzyme was significantly larger than that formed from the FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS Reactivase for coenzyme B12-dependent enzyme K Mori et al enzymCN-Cbl complex These results are consistent with previous data [17] Three bands were clearly seen in the lanes containing the free reactivase, i.e two adjacent thick bands and one thin band on the front line, irrespective of the presence of enzyme (Fig 5A, lanes b, c, e, and g) To determine their subunit compositions, bands i–vi were excised and subjected to SDS ⁄ PAGE on 6% and 14% gels (Fig 5B,C), followed by densitometric analyses Both band i and band vi of the enzymreactivase complexes comprised all of the subunits from the enzyme (a, b, and c) and the reactivase (a and b) (Fig 5B,C, lanes i and vi) Subunit compositions of the bands were the same when either the enzymCN-Cbl complex or apoenzyme was used (data not shown) If the a, b and c subunits of the enzyme are abbreviated as aD, bD, and cD, respectively, and the a and b subunits of the reactivase are abbreviated as aR and bR, respectively, molar ratios of aD, bD, cD, aR and bR in bands i and vi were determined to be about : : : : and : : : : 1, respectively, by densitometric analysis Therefore, it was demonstrated that bands i and vi are (aDbDcD)2Ỉ(aRỈaRbR) and (aDbDcD)2Ỉ(aRỈaRbR)2 complexes, respectively We named the former the enzymreactivase (1 : 1) complex and the latter the enzymreactivase (1 : 2) complex When bands ii and iii of Fig 5A, i.e two thick bands of the reactivase, were subjected to SDS ⁄ PAGE, they contained both aR and bR, although the ratios of subunits were different (Fig 5C, lanes ii and iii) Densitometric analysis indicated that molar ratios of aR to bR for bands ii and iii were approximately : and : 1, respectively These results indicated that the upper and lower bands of the reactivase correspond to the aRỈaRbR and (aRbR)2 complexes, respectively The thin band on the front line of nondenaturing PAGE (Fig 5A, band iv) contained only bR (Fig 5C, lane iv) It possibly represents a monomer of bR, as there is no direct interaction between two adjacent bR subunits in the crystal structure of the (aRbR)2 tetramer of the reactivase [25] In the absence of ADP ⁄ Mg2+ or in the presence of ADP but in the absence of Mg2+, the enzymreactivase complex was formed in small amounts from apoenzyme and the reactivase and not at all from the enzymCN-Cbl complex and the reactivase (data not shown) Affinity of the reactivase for DD Figure shows the dependence of enzymreactivase complex formation on reactivase concentration The A Fig Dependence of complex formation on reactivase concentration at a fixed enzyme concentration (A) Apoenzyme + reactivase (left, none; right, +ADP) (B) EnzymCN-Cbl complex + reactivase (left, none; right, +ADP) Experimental conditions were similar to those for nondenaturing PAGE in Fig 5, except that the reactivase and enzyme concentrations were varied and fixed (1 lM), respectively The number on the top of each lane indicates the reactivase concentration (lM) Lane R: reactivase lM Positions of the enzyme, reactivase and the enzymreactivase complexes are indicated on the right of the gels: (i) enzymreactivase (1 : 2) complex; (ii) enzymreactivase (1 : 1) complex; (iii) enzyme; (iv) reactivase (aRỈaRbR); (v) reactivase [(aRbR)2]; (vi) small subunit of the reactivase (bR) B FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS 4937 Reactivase for coenzyme B12-dependent enzyme K Mori et al enzymreactivase (1 : 1) complex (band ii) was formed even at the lowest concentration of reactivase tested (0.25 lm) in the presence of ADP and Mg2+ In the absence of ADP and Mg2+, it was observable at ‡ 0.5 lm reactivase Similarly, the enzymreactivase (1 : 2) complex (band i) appeared clearly at lm reactivase in the presence of ADP and Mg2+, whereas it was observed at ‡ lm reactivase in the absence of ADP and Mg2+ Moreover, although the enzymreactivase (1 : 2) species was the only enzymreactivase complex observed at ‡ lm reactivase in the presence of ADP and Mg2+, some enzymreactivase (1 : 1) complex remained even at the highest concentration of reactivase tested (20 lm) in the absence of ADP and Mg2+ The apparent KD values of the reactivase for formation of the enzymreactivase complex were 0.4 lm and lm in the presence and absence of ADP and Mg2+, respectively When similar experiments were carried out with the enzymCN-Cbl complex in place of apoenzyme, essentially no complex formation was observed, even at 20 lm reactivase, in the absence of ADP and Mg2+ In contrast, in the presence of ADP and Mg2+, the reactivase formed complexes with DD, accompanying the release of tightly bound CN-Cbl from the enzyme [17] However, the enzymreactivase (1 : 1) complex remained at 20 lm reactivase The apparent KD of the reactivase was 0.7 lm Discussion In the present study, we demonstrated the multiple turnovers of the DD-reactivating factor in the in vitro activation of the inactive enzymCN-Cbl complex, and thus redesignated the reactivating factor [16,17] DD reactivase This is reasonable in vivo from the viewpoint of the cellular economy of energy If the reactivating factor could not mediate the multiple exchanges in the reactivation of inactivated holoenzymes, its presence would not be advantageous to the bacterial cells We have previously demonstrated that the hydrolysis of ATP by the reactivating factor is catalytic [17] Moreover, the reactivation observed in permeabilized cells of K oxytoca seems to be catalytic [13,14], although molar ratios of the reactivating factor to the enzyme remain obscure We previously failed to demonstrate the multiple turnovers of the reactivase in the reactivation of inactivated holoenzymes One possible reason for this difficulty might be the accelerated rate of inactivation of the enzyme with b-hydroxypropionaldehyde accumulating to an extremely high concentration (20–180 mm), as the reactivation was monitored by product formation from reactivated holoenzyme at high concentrations of the enzyme and 4938 the reactivase It would be easier to demonstrate the multiple turnovers of DD reactivase and glycerol dehydratase reactivase in the in situ reactivation, because the reactivation takes place in toluene-treated cells, where local concentrations of the enzyme and the reactivase are high enough for reactivation, and an inhibitory product, b-hydropropionaldehyde, diffuses away From the initial rate of exchange of enzyme-bound CN-Cbl for AdePeCbl, the rate constant of the reactivase in cobalamin release (kcat,cbl-release) for CN-Cbl was calculated to be 0.27 min)1 at 37 °C From the initial rate of reactivation of the glycerol-inactivated holoenzyme, the rate constant of the reactivase in the reactivation (kcat,react) was calculated to be 0.071 ± 0.008 min)1 at 37 °C Considering that the enzyme contains two cobalamin-binding sites in the (abc)2 dimer, it can be assumed that the reactivase mediates the exchange of enzyme-bound damaged cofactor for intact AdoCbl with a rate constant (kcat,cbl-release) of 0.14 min)1 This value is about half of the above-mentioned kcat,cbl-release for CN-Cbl release This difference might be attributable to the difference in release rate between CN-Cbl and the damaged cofactor Another possible explanation is that the enzyme activity of the reconstituted abcỈAdoCbl complex in one trimer might be affected by the neighboring trimer of the same enzyme molecule, i.e by the presence of the damaged cofactor or AdoCbl and their absence The kcat of the reactivase in ATP hydrolysis in the presence of enzyme (1.2 min)1) was slightly smaller than that in its absence (1.4 min)1) It was in the same range as the rate constant of the enzyme in the suicide inactivation with glycerol (1.3 min)1) [17], but about five-fold and 10-fold larger than the rate constants for the release of CN-Cbl (0.27 min)1) and the damaged cofactor (0.14 min)1), respectively Therefore, ATP hydrolysis and cobalamin release or reactivation might be not very tightly coupled The reactivation of the inactivated holoenzymes by the reactivase seems to be physiologically relevant, because kcat,cbl-release is much larger than the rate constant for bacterial growth on glycerol The reactivase exhibited broad specificities for nucleotides and divalent metal cations, both of which are absolutely required for the in vitro activation of the enzymCN-Cbl complex We have previously reported similar specificities in the in situ reactivation of the glycerol-inactivated hologlycerol dehydratase with K pneumoniae cells [13] It was established that the reactivase-mediated reactivation of the inactivated holoenzymes with ATP and Mg2+ takes place in two steps: (a) ADP-dependent cobalamin release with FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS K Mori et al concomitant formation of the apoenzymreactivase complex; and (b) ATP-dependent dissociation of the complex to apoenzyme and the reactivase [17] ATP plays dual roles, i.e as a precursor of ADP in the first step, and an effector to change the reactivase to a form with low affinity for the enzyme The nucleotides used in this study (ATP, GTP, CTP, UTP, 2¢-dATP, and 3¢dATP) were effective in overall activation, although the efficiencies were somewhat different This suggests that these nucleotides are also effective in both steps We determined the crystal structure of the reactivase in the ADP-bound and nucleotide-free forms [25] ADP is bound to the ATPase domain, a core domain of the a subunit This domain shares common structural features with the ATPase domain of actin superfamily proteins, including Hsp70 molecular chaperones The reactivase binds ADP without specific interactions with the adenine ring through hydrogen bonding or base stacking The broad specificity of the reactivase for the base moiety is thus consistent with its crystal structure The O2¢ atom of ADP is hydrogen bonded to the –COO) group of Glua459 and the e-NH2 group of Lysa462 These hydrogen bonds exist in the interaction between Hsc70 and ADP as well The residue corresponding to Glua459 of the DD reactivase is Alaa461 in glycerol dehydratase reactivase Furthermore, 2¢-dATP retained half of the efficacy of ATP in the activation of the enzymCN-Cbl complex It was therefore concluded that these hydrogen bonds are not essential for (re)activation Similarly, 3¢-dATP retained half of the efficacy of ATP in the activation In the crystal structure of the reactivase, no amino acids were found to be hydrogen bonded to O3¢ of ADP Thus, no requirement for the 3¢-OH group seems to be reasonable from its crystal structure The reactivase has two distinct divalent metal ionbinding sites in the ab heterodimeric unit [25] One of them is present in the interface between the a and b subunits This metal ion is coordinated by four amino acids (Aspa166, Aspa183, Thra105, and Glub31), all of which are completely conserved in both reactivases for diol and glycerol dehydratases These coordinations are maintained in the reactivase, irrespective of the ADP binding The crystal structure of the DD reactivase suggested that this metal ion is Mg2+ in the ADP-bound form, whereas it is Ca2+ in the nucleotide-free form It might be possible that Mg2+ occupies this site in vivo and is replaced by Ca2+ in the purification of the reactivase by hydroxyapatite column chromatography [25] Liao et al also reported this metal ion to be Ca2+ in the nucleotidefree form of glycerol dehydratase reactivase that is crystallized in the presence of Ca2+ [24] In the case of Reactivase for coenzyme B12-dependent enzyme the ADP-bound form, Ca2+ in this site may be replaced by Mg2+ upon incubation of the reactivase with ADP and Mg2+ The other divalent metal ion is Mg2+, which interacts with the b-phosphate group of ADP in the nucleotide-binding site of the a subunit This Mg2+ was not found in the nucleotide-free form Mg2+, Mn2+, Co2+ and Ni2+ enhanced the ATPase activity of the reactivase, although the reactivase can hydrolyze ATP to ADP even without divalent metal ions These metal ions were effective in the activation of the enzymCN-Cbl complex by the reactivase in the presence of ATP, although relative efficiencies were not always correlated On the other hand, the reactivase was unable to activate the enzymCN-Cbl complex even in the presence of ATP with Ca2+, Cr2+or Cu2+ or without divalent metal ions These metal ions had little or no enhancing effect on the ATPase activity of the reactivase Thus, the reactivase-mediated activation of the enzymCN-Cbl complex absolutely requires the hydrolysis of ATP in the presence of divalent metal ions The reactivase does not form the enzymreactivase complexes from the enzymCN-Cbl complex in the presence of ADP without divalent cations These results suggest that the binding of ADP alone to the ATPase domain of the reactivase a subunit is not sufficient to cause a conformational change of the enzyme, resulting in the release of adenine-lacking cobalamins, such as CN-Cbl and damaged cofactor The fact that the relative efficiencies of metal ions for the reactivation are not always correlated with the ATPase activity of the reactivase might be attributable to the different characteristics of binding of these divalent ions to the other metal ion-binding site in the interface between the a and b subunits, although the binding specificity of this site for metal ions remains unclear We have previously demonstrated the formation of the enzymreactivase complex from apoenzyme and the reactivase in the presence of ADP ⁄ Mg2+ or in the absence of nucleotide, although the exact subunit compositions of the resulting complexes remained unclear Our present study indicated that two kinds of complexes with different subunit compositions were formed These complexes contain the enzyme and the reactivase in : and : molar ratios, and release of the reactivase b subunit was observed upon complex formation (Fig 7A) These results constitute clear evidence for the displacement of the reactivase b subunit by the enzyme b subunit (subunit swapping) upon formation of the complex between the enzyme and the reactivase At present, it is not clear which complex is involved or whether both complexes are involved in the reactivation The dissociation of (aRbR)2 into FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS 4939 Reactivase for coenzyme B12-dependent enzyme K Mori et al cofactor to pass through it Intact cofactor, an adenine-containing cobalamin, is not released from the enzyme by the reactivase One reason might be its larger size, and the other possible reason might be that the additional interaction between its adenine moiety and the enzymes’s adenine-binding pocket stabilizes the interaction between the enzyme a and b subunits In contrast, even in the absence of ADP, the reactivase forms the enzymreactivase complex with apoenzyme However, it does not release the damaged cofactor from the inactivated holoenzymes under this condition This may be because the steric repulsion is less or is canceled by the conformational flexibility in the absence of ADP ⁄ Mg2+ In order to prove or disprove these predictions, we have to await the structural analysis of a real enzymreactivase complex A B Experimental procedures Fig Subunit swapping between DD and the reactivase (DD-R) (A) and the existence of a cavity between DD a (pink) and b (green) subunits (B) a, aD (pink) or aR (light blue) subunit; b, bD (green) or bR (orange) subunit; c, cD subunit (dark blue) aRỈaRbR and bR in the presence of ADP and Mg2+ was observed even without the enzyme The crystal structure of the reactivase also suggested that the interactions between the reactivase a and b subunits are weakened at least partially by the ADP binding [25] The space that is opened by the dissociation of the reactivase b subunit would most likely be occupied by the enzyme b subunit, as these subunits have similar folds [25,28] The docking model of the aDbDcDỈaR complex indicates that marked steric repulsion is induced between the enzyme a subunit and the reactivase a subunit in the complex [25] The amino acid side chains that come closer than the van der Waals contact in the modeled structure would push each other aside and result in tilting of the enzyme a subunit with respect to the enzyme b subunit Thus, it would lead to the release of the damaged cofactor, an adenine-lacking cobalamin, from the enzyme, because cobalamin is bound between the a and b subunits of the enzyme The crystal structure of DD revealed that, like ethanolamine ammonia lyase [29], the enzyme has ˚ ˚ a cavity  A in height and  15 A in width between the a and b subunits (Fig 7B) The tilting of the a subunit with respect to the b subunit upon subunit ˚ swapping is estimated to be  A, based on the mod˚ eled complex, forming a cavity  11 A in height The size of this cavity is comparable with that of adeninelacking cobalamins, and thus allows the damaged 4940 Materials Crystalline AdoCbl was a gift from Eisai (Tokyo, Japan) CN-Cbl was obtained from Glaxo Research Laboratories (Greenford, UK) AdePeCbl was synthesized according to published procedures [30] [32P]ATP[cP] was obtained from PerkinElmer (Waltham, MA, USA) 2¢-DeoxyATP and 3¢-deoxyATP were obtained from Sigma-Aldrich (St Louis, MO, USA) All other chemicals were commercial products of the highest grade available and were used without further purification K oxytoca recombinant DD and its reactivase were purified to homogeneity from overexpressing Escherichia coli JM109 harboring expression plasmid pUSI2E(DD) [31] and E coli JM109 or B834 harboring expression plasmid pUSI2ENd(6 ⁄ 5b) [16,32], respectively, as reported previously Enzyme and protein assays The amount of aldehydic products formed by DD was determined by the 3-methyl-2-benzothiazolinone hydrazone method [33] One unit of the enzyme is defined as the amount of enzyme activity that catalyzes the formation of lmol of propionaldehyde per minute at 37 °C The reactivation of the glycerol-inactivated holoenzyme and the activation of the enzymCN-Cbl complex by the reactivase were assayed with 1,2-propanediol as substrate in the presence of 21 lm AdoCbl and appropriate concentrations of ATP and MgCl2 In some experiments, ATP and MgCl2 were replaced with other nucleotides and chloride salts of divalent metal cations, respectively The protein concentrations of the purified enzyme and reactivase were determined by measuring the absorbance at 280 nm, based on the method of Gill and von Hippel [34], as described previously [16] FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS K Mori et al Glycerol-inactivated holoenzyme and enzymCN-Cbl complex The glycerol-inactivated holoenzyme was formed by incubation of apoenzyme (70 units, 2.8 nmol) with 38 lm AdoCbl at 37 °C for 30 in 1.7 mL of 0.05 m potassium phosphate buffer (pH 8.0) containing 19% glycerol Glycerol and excess AdoCbl were removed by dialysis at °C for 49 h against 600 volumes of 0.05 m potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol with a buffer change The enzymCN-Cbl complex was prepared by incubation of apoenzyme (30–45 units, 1.2–1.8 nmol) with 13 lm CN-Cbl at 37 °C for 30 in 540 lL of 0.05 m potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol and 0.6–1% Brij35 ATPase activity The ATP-hydrolyzing activity of the reactivase was assayed by the release of [32P]Pi from [32P]ATP[cP], as described previously [17], with minor modifications Appropriate concentrations of [32P]ATP[cP] ( · 103 d.p.m per nmol) and MgCl2 were incubated at 37 °C for appropriate times with the reactivase, in the presence and absence of apoenzyme, in 0.01 m potassium phosphate buffer (pH 8.0) in a total volume of 50 lL After termination of the reaction by addition of 0.45 mL of an ice-cold suspension of 6% (w ⁄ v) charcoal in 50 mm NaH2PO4 and mixing vigorously for 10 min, the charcoal was removed by centrifugation at 16 000 g for The amount of radioactivity in 0.2 mL of the supernatant was determined by liquid scintillation counting, and ATPase activity was obtained by subtracting the radioactivity of a minus reactivase control In some experiments, MgCl2 was replaced with other divalent metal chlorides Reactivase-mediated exchange of enzyme-bound CN-Cbl for AdePeCbl The enzymCN-Cbl complex was formed by incubation of 30 units of apoenzyme (1.2 nmol) with 80 lm CN-Cbl at 37 °C for 60 in 75 lL of 0.04 m potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol and 0.5% Brij35 To the resulting mixture were added 19 lg of reactivase (0.12 nmol), together with 40 lm AdePeCbl, 20 mm ATP and 20 mm MgCl2 in 0.03 m potassium phosphate buffer (pH 8.0) containing 1% 1,2-propanediol and 0.1% Brij35, in a total volume of 150 lL After incubation at 37 °C for appropriate periods, the exchange reaction was terminated by addition of 150 lL of 0.01 m potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol, 0.2% Brij35, and 60 mm EDTA The resulting mixture was then subjected to ultrafiltration on a Microcon YM-10 microconcentrator (Millipore, Billerica, MA, USA) to remove unbound cobalamins The protein fraction retained on the filter was washed twice by the addition of 150 lL of Reactivase for coenzyme B12-dependent enzyme 0.01 m potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol, 0.2% Brij35, and 10 mm EDTA, and this was followed by ultrafiltration The spectrum was measured after addition of 150 lL of 0.01 m potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol, 0.2% Brij35 and 10 mm EDTA to the protein fraction Experiments without the reactivase or ATP ⁄ MgCl2 were also performed as controls Similar experiments without cobalamins were also carried out, for correction of spectra After spectral measurement, 200 lL of the mixture was incubated at 37 °C for 10 with 0.06 m citric acid and m guanidine hydrochloride to denature the proteins After neutralization, mg of KCN was added, and the resulting mixture (440 lL) was illuminated for 10 on ice with a 250 W tungsten light bulb from a distance of 10 cm to convert cobalamins to a dicyano form The total amount of enzymebound cobalamin was calculated from the absorbance at 368 nm for dicyanocobalamin (eM, 368, 30.4 · 103 m)1Ỉcm)1) [35] Analysis of the enzymreactivase complex by gel filtration Complex formation between DD and the reactivase was analyzed by gel filtration on a Superose (10 ⁄ 300 GL) column, using an FPLC system (GE Healthcare, Little Chalfont, UK) In the presence and absence of 21 mm adenine nucleotide (ATP or ADP) and 21 mm MgCl2, apoenzyme (12 units, 0.48 nmol) were incubated at 37 °C for 60 with 0.27 mg (1.7 nmol) of reactivase in 170 lL of 0.04 m potassium phosphate buffer (pH 8.0) containing 1.4% 1,2-propanediol and 0.7% Brij35 The resulting mixture was applied to a column that had been equilibrated with 0.05 m potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol and 0.5% Brij35 with or without the corresponding adenine nucleotide (ADP or ATP) and MgCl2 (1 mm each) The column was developed with the same buffer at a flow rate of 0.4 mLỈmin)1 The enzyme and the reactivase alone were also applied under the same conditions as controls The elution of proteins was monitored by the absorbance at 280 nm Analysis of the enzymreactivase complexes by PAGE The reactivase (12 lg, 76 pmol) was incubated with 10 mm dithiothreitol at 30 °C for 30 in lL of 0.01 m potassium phosphate buffer (pH 8.0) ADP and MgCl2 (10 mm each) were added to the resulting mixture in a total volume of lL, and the mixture was incubated at 20 °C for 20 to form the ADP-bound form of the reactivase The enzymCN-Cbl complex was formed by the incubation of apoenzyme (0.38 units, 15 pmol) with 15 lm CN-Cbl at 30 °C for 30 in lL of 0.01 m potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol and FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS 4941 Reactivase for coenzyme B12-dependent enzyme K Mori et al 1% Brij35 Six microliters of the mixture containing the reactivasADP complex were added to lL of the mixture containing the enzymCN-Cbl complex After 1.5 h at 30 °C, the mixture was subjected to PAGE on a 5% gel under nondenaturing conditions, as described by Davis [36], with some modifications: 0.1 m 1,2-propanediol, mm ADP, mm MgCl2 and mm dithiothreitol were added to the gels, and 0.1 m 1,2-propanediol, mm ADP and mm Mg(OCOCH3)2 were added to the electrode buffer The enzymCN-Cbl complex, apoenzyme and the reactivasADP complex alone were also subjected to electrophoresis under the same conditions After electrophoresis, protein was stained with Coomassie Brilliant Blue R-250 Bands were excised from the gel, soaked in water, and equilibrated with the SDS-containing sample buffer Then excised gels were subjected to SDS ⁄ PAGE on 14% and 6% gels under the conditions described by Laemmli [37] Protein was stained again with Coomassie Brilliant Blue R-250 Densitometric analysis of gels was performed with a Printgraph AE-6911CX system (ATTO, Tokyo, Japan) and nih-image, version 1.6.3 (National Institutes of Health) Model figure Figure 7B was generated with chimera [38], using the atomic coordinates for the enzymCN-Cbl complex (Protein Data Bank accession code: 1EGM) Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research [(B) 13480195 and 17370038 and Priority Areas 753 and 513 to T Toraya], a Grant-inAid for Young Scientists [(B) 18770111 to K Mori] from the Japan Society for Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a Grant in Aid for Natural Sciences Research (to T Toraya) from the Asahi Glass Foundation, Tokyo, Japan We thank Y Kurimoto for her assistance with manuscript preparation References Banerjee R, ed (1999) Chemistry and Biochemistry of B12 Wiley, New York, NY ´ Retey J (1990) Enzymic reaction selectivity by negative catalysis or how enzymes deal with highly reactive intermediates? Angew Chem Int Ed Engl 29, 355–361 Toraya T (2003) Radical catalysis in coenzyme B12-dependent isomerization (eliminating) reactions Chem Rev 103, 2095–2127 Lee HA Jr & Abeles RH (1963) Purification and properties of dioldehydrase, an enzyme requiring a cobamide coenzyme J Biol Chem 238, 2367–2373 4942 Toraya T, Shirakashi T, Kosuga T & Fukui S (1976) Substrate specificity of coenzyme B12-dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator Biochem Biophys Res Commun 69, 475–481 Toraya T (1994) Diol dehydratase and glycerol dehydratase, coenzyme B12-dependent isozymes In Metal Ions in Biological Systems (Sigel H & Sigel A eds), Vol 30, pp 217–254 Marcel Dekker, New York, NY Toraya T & Fukui S (1982) Diol dehydratase In B12 (Dolphin D ed), Vol 2, pp 233–262 Wiley, New York, NY Toraya T, Honda S, Kuno S & Fukui S (1978) Coenzyme B12-dependent diol dehydratase: regulation of apoenzyme synthesis in Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 J Bacteriol 135, 726–729 Toraya T, Kuno S & Fukui S (1980) Distribution of coenzyme B12-dependent diol dehydratase and glycerol dehydratase in selected genera of Enterobacteriaceae and Propionibacteriaceae J Bacteriol 141, 1439–1442 10 Bachovchin WW, Eagar RG Jr, Moore KW & Richards JH (1977) Mechanism of action of adenosylcobalamin: glycerol and other substrate analogues as substrates and inactivators for propanediol dehydratase – kinetics, stereospecificity, and mechanism Biochemistry 16, 1082–1092 11 Schneider Z & Pawelkiewicz J (1966) The properties of glycerol dehydratase isolated from Aerobacter aerogenes, and the properties of the apoenzyme subunits Acta Biochim Pol 13, 311–328 12 Poznanskaya AA, Yakusheva MI & Yakovlev VA (1977) Study of the mechanism of action of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes II The inactivation kinetics of glycerol dehydratase complexes with adenosylcobalamin and its analogs Biochim Biophys Acta 484, 236–243 13 Honda S, Toraya T & Fukui S (1980) In situ reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase J Bacteriol 143, 1458–1465 14 Ushio K, Honda S, Toraya T & Fukui S (1982) The mechanism of in situ reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase J Nutr Sci Vitaminol 28, 225–236 15 Mori K, Tobimatsu T, Hara T & Toraya T (1997) Characterization, sequencing, and expression of the genes encoding a reactivating factor for glycerolinactivated adenosylcobalamin-dependent diol dehydratase J Biol Chem 272, 32034–32041 16 Toraya T & Mori K (1999) A reactivating factor for coenzyme B12-dependent diol dehydratase J Biol Chem 274, 3372–3377 17 Mori K & Toraya T (1999) Mechanism of reactivation of coenzyme B12-dependent diol dehydratase by a FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS K Mori et al 18 19 20 21 22 23 24 25 26 27 molecular chaperone-like reactivating factor Biochemistry 38, 13170–13178 Tobimatsu T, Kajiura H, Yunoki M, Azuma M & Toraya T (1999) Identification and expression of the genes encoding a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase J Bacteriol 181, 4110–4113 Kajiura H, Mori K, Tobimatsu T & Toraya T (2001) Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase J Biol Chem 276, 36514–36519 Seifert C, Bowien S, Gottschalk G & Daniel R (2001) Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii Eur J Biochem 268, 2369–2378 ´ Pierik AJ, Graf T, Pemberton L, Golding BT & Retey J (2008) But-3-ene-1,2-diol: a mechanism-based active site inhibitor for coenzyme B12-dependent glycerol dehydratase Chembiochem 9, 2268–2275 Toraya T, Tamura N, Watanabe T, Yamanishi M, Hieda N & Mori K (2008) Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol J Biochem 144, 437–446 Kajiura H, Mori K, Shibata N & Toraya T (2007) Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases FEBS J 274, 5556–5566 Liao D-I, Reiss L, Turner I Jr & Dotson G (2003) Structure of glycerol dehydratase reactivase: a new type of molecular chaperone Structure 11, 109–119 Shibata N, Mori K, Hieda N, Higuchi Y, Yamanishi M & Toraya T (2005) Release of a damaged cofactor from a coenzyme B12-dependent enzyme: X-ray structures of diol dehydratase-reactivating factor Structure 13, 1745– 1754 Mori K, Bando R, Hieda N & Toraya T (2004) Identification of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase J Bacteriol 186, 6845–6854 Baker JJ & Stadiman TC (1982) Amino mutases In B12 (Dolphin D ed), Vol 2, pp 203–232 Wiley, New York, NY Reactivase for coenzyme B12-dependent enzyme 28 Shibata N, Masuda J, Tobimatsu T, Toraya T, Suto K, Morimoto Y & Yasuoka N (1999) A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase Structure 7, 997–1008 29 Shibata N, Tamagaki H, Hieda N, Akita K, Komori H, Shomura Y, Terawaki S, Mori K, Yasuoka N, Higuchi Y & Torayo T (2010) Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates J Biol Chem 285, 26484–26493 30 Sando GN, Grant ME & Hogenkamp HPC (1976) The interaction of adeninylalkylcobalamins with ribonucleotide reductase Biochim Biophys Acta 428, 228–232 31 Tobimatsu T, Sakai T, Hashida Y, Mizoguchi N, Miyoshi S & Toraya T (1997) Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca Arch Biochem Biophys 347, 132–140 32 Mori K, Hieda N, Yamanishi M, Shibata N & Toraya T (2005) Crystallization and preliminary X-ray analysis of molecular chaperone-like diol dehydratasereactivating factor in ADP-bound and nucleotide-free forms Acta Crystallogr F 61, 603–605 33 Toraya T, Ushio K, Fukui S & Hogenkamp HPC (1977) Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme J Biol Chem 252, 963–970 34 Gill SC & von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data Anal Biochem 182, 319–326 35 Barker HA, Smyth RD, Weissbach H, Munch-Petersen A, Toohey JI, Ladd JN, Volcani BE & Wilson RM (1960) Assay, purification, and properties of the adenylcobamide coenzyme J Biol Chem 235, 181–190 36 Davis BJ (1964) Disc electrophoresis II Method and application to human serum proteins Ann NY Acad Sci 121, 404–427 37 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 38 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC & Ferrin TE (2004) UCSF Chimera – a visualization system for exploratory research and analysis J Comput Chem 25, 1605–1612 FEBS Journal 277 (2010) 4931–4943 ª 2010 The Authors Journal compilation ª 2010 FEBS 4943 ... Hieda N, Yamanishi M, Shibata N & Toraya T (2005) Crystallization and preliminary X-ray analysis of molecular chaperone-like diol dehydratasereactivating factor in ADP-bound and nucleotide-free forms... reactivating factors – that is, subunit swapping might occur However, no biochemical evidence for this has been obtained so far A similar reactivating factor for ethanolamine ammonia lyase has been... we have to await the structural analysis of a real enzymreactivase complex A B Experimental procedures Fig Subunit swapping between DD and the reactivase (DD-R) (A) and the existence of a cavity